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Anal. Chem. 2009, 81, 2944–2952

Gel Electrophoresis Method to Measure the Concentration of Single-Walled Carbon Nanotubes Extracted from Biological Tissue Ruhung Wang,† Carole Mikoryak,† Elena Chen,† Synyoung Li,† Paul Pantano,‡,§ and Rockford K. Draper*,†,‡,§

Anal. Chem. 2009.81:2944-2952. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 07/24/18. For personal use only.

Department of Molecular and Cell Biology, Department of Chemistry, and The Alan G. MacDiarmid NanoTech Institute, The University of Texas at Dallas, Richardson, Texas 75080 A rapid and sensitive method to detect single-walled carbon nanotubes (SWNTs) in biological samples is presented. The method uses polyacrylamide gel electrophoresis (PAGE) followed by quantification of SWNT bands. SWNTs dispersed in bovine serum albumin (BSA) were used to develop the method. When BSA-SWNT dispersions were subjected to sodium dodecyl sulfate (SDS)-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected. The SWNTs, however, accumulated in a sharp band at the interface between the loading well and the stacking gel. The intensities from digitized images of these bands were proportional to the amount of SWNTs loaded onto the gel with a detection limit of 5 ng of SWNTs. To test the method, normal rat kidney (NRK) cells in culture were allowed to take up SWNTs upon exposure to medium containing various concentrations of BSA-SWNTs for different times and temperatures. The SDS-PAGE analyses of cell lysate samples suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis at a rate of 30 fg/day/cell upon exposure to medium containing 98 µg/mL SWNTs. The list of potential applications for single-walled carbon nanotubes (SWNTs) is diverse and extends across boundaries of physical and biological sciences. Physical applications include nanoelectronics, energy storage, enhancing composite materials, field-emission devices, and chemical sensors.1,2 Biological applications include biosensors, drug delivery, antimicrobial treatments, and cancer chemotherapy.3-5 An often underappreciated challenge in many of these applications is simply measuring the amount of SWNTs in liquid samples. Compounding this challenge * To whom correspondence should be addressed. E-mail: draper@ utdallas.edu. Fax: 972-883-2409. † Department of Molecular and Cell Biology. ‡ Department of Chemistry. § The Alan G. MacDiarmid NanoTech Institute. (1) Avouris, P.; Chen, Z.; Perebeinos, V. Nat. Nanotechnol. 2007, 2, 605–615. (2) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Science 2002, 297, 787– 792. (3) Prato, M.; Kostarelos, K.; Bianco, A. Acc. Chem. Res. 2008, 41, 60–68. (4) Lacerda, L.; Bianco, A.; Prato, M.; Kostarelos, K. Adv. Drug Delivery Rev. 2006, 58, 1460–1470. (5) Lin, Y.; Taylor, S.; Li, H.; Fernando, K. A. S.; Qu, L.; Wang, W.; Gu, L.; Zhou, B.; Sun, Y.-P. J. Mater. Chem. 2004, 14, 527–541.

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are the facts that most commercially supplied SWNT powders contain a mixture of SWNT types plus contaminants of residual metal catalysts and nontubular carbon (NTC) species. Measurement priorities and protocols for working with SWNT powders were the topics of two joint NASA/NIST conferences and resulted in a recently published practical guide documenting the use of multiple approaches for quantifying and characterizing SWNT powders, including thermogravimentric analysis, near-infrared spectroscopy, Raman spectroscopy, and various high-resolution imaging methods.6 Nonetheless, there remains a need for rapid and sensitive methods to quantify small amounts of SWNTs in liquid samples, especially in biological applications. For example, microprobe Raman spectroscopy7,8 and the intrinsic near-infrared photoluminescence of SWNTs themselves8-13 have been used to detect SWNTs within single cells, but the methods are not well suited to quantify SWNTs from populations of cells. Moreover, microprobe Raman spectroscopy and near-infrared spectroscopy require expensive instrumentation. In addition, other reported methods require either the attachment of a fluorescent or radioactive label to SWNTs, which can potentially create problems by modifying the properties of the SWNTs, or if the label comes off. In this paper, we describe and document the use of polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) for quantifying small amounts of SWNTs. We observed that SWNTs dispersed in a typical SDS sample buffer migrate toward the anode in a PAGE apparatus to become trapped and concentrated at the interface between the sample loading well and the stacking gel where they appear as a dark band. Raman (6) Freiman, S., Hooker, S., Migler, K., Arepalli, S., Eds. Measurement Issues in Single Wall Carbon Nanotubes; NIST Recommended Practice Guide, Special Publication 960-19; NIST: Gaithersburg, MD, 2008. (7) Yehia, H. N.; Draper, R. K.; Mikoryak, C.; Walker, E. K.; Bajaj, P.; Musselman, I. H.; Daigrepont, M. C.; Dieckmann, G. R.; Pantano, P. J. Nanobiotechnol. 2007, 5, 8–24. (8) Heller, D. A.; Baik, S.; Eurell, T. E.; Strano, M. S. Adv. Mater. 2005, 17, 2793–2799. (9) Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. J. Am. Chem. Soc. 2004, 126, 15638–15639. (10) Heller, D. A.; Jeng, E. S.; Yeung, T.-K.; Martinez, B. M.; Moll, A. E.; Gastala, J. B.; Strano, M. S. Science 2006, 311, 508–511. (11) Jin, H.; Heller, D. A.; Strano, M. S. Nano Lett. 2008, 8, 1577–1585. (12) Welsher, K.; Liu, Z.; Daranciang, D.; Dai, H. Nano Lett. 2008, 8, 586–590. (13) Cherukuri, P.; Gannon, C. J.; Leeuw, T. K.; Schmidt, H. K.; Smalley, R. E.; Curley, S. A.; Weisman, R. B. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18882–18886. 10.1021/ac802485n CCC: $40.75  2009 American Chemical Society Published on Web 03/18/2009

spectroscopy confirmed that all detectable SWNTs in the applied sample were recovered in the band. A common flatbed scanner was used to digitize and measure the band intensities. Comparison with calibrated standards enabled rapid and reliable estimation of SWNT amounts present in small volumes (1-60 µL) without need for expensive instrumentation. We have used this SDS-PAGE method to quantify the uptake of bovine serum albumin (BSA)coated SWNTs by cultured normal rat kidney (NRK) cells. In these experiments, NRK cells were exposed to media containing various concentrations of SWNTs for different times and temperatures before being collected, lysed, and subjected to SDS-PAGE analyses. SWNT uptake was linear with respect to time and BSA-SWNT concentration and did not occur at 4 °C, which is consistent with an uptake model involving fluid-phase endocytosis. EXPERIMENTAL SECTION Materials and Solutions. The CoMoCAT SWNT-containing powder (lot no. UT4-A001) was purchased from SouthWest NanoTechnologies (Norman, OK). Caution: a particulates respirator should be worn when handling dry SWNT powders. Dulbecco’s modified Eagle medium (DMEM) and trypsin were purchased from Irvine Scientific (Santa Ana, CA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT). Aqueous BSA (SigmaAldrich, St. Louis, MO) solutions (12.5-200 mg/mL) were filtered through a 0.2 µm membrane and stored at 4 °C. Phosphatebuffered saline (PBS; 8 mM phosphate, 150 mM NaCl, pH 7.4) was sterilized by autoclaving at 120 °C for 0.5 h. Deionized water (18.3 MΩ · cm) was obtained using a Nanopure Infinity water purification system (Barnstead, Dubuque, IA). All other chemicals were purchased from Sigma-Aldrich and were used as received. BSA-SWNT Standards and Dispersions. A standard 1.0 mg/mL BSA-SWNT suspension was prepared by dispensing 1.0 mg of the as-received CoMoCAT SWNT powder into a 1.5 mL microcentrifuge tube. An amount of 1.0 mL of 100 mg/mL aqueous BSA was added and probe-sonicated at 0 °C for 10 min using a Branson 250 sonifier operated at 12 W. Probe sonication was performed using a 60 s “on”/10 s “off” pulse sequence with a 3 mm diameter probe tip that was placed two-thirds of the distance below the surface of the suspension. The BSA-SWNT standard was used directly as a calibrated solution that had a known weight of SWNT powder in a known volume. BSA-SWNT dispersions were prepared using a centrifugation protocol designed to remove heavy impurities such as metal-containing SWNTs and bundles.7 Specifically, BSA-SWNT suspensions prepared using 1.0 mg/mL SWNTs and 12.5-200 mg/mL BSA were centrifuged in an Eppendorf 5417C centrifuge for 2 min at 16 000g. The upper 900 µL of this first supernatant was recovered without disturbing the sediment and placed in a clean tube before a second 2 min centrifugation at 16 000g was performed. The upper 850 µL of this second supernatant was carefully recovered to generate a BSA-SWNT dispersion. In some cases, individual dispersions were pooled and stored at 4 °C for later use; all dispersions could be stored for at least 1 month without any SWNTs precipitating out of solution. Absorption Spectroscopy. Absorption spectra of BSA-SWNT dispersions were acquired using a dual-beam Perkin-Elmer Lambda 900 UV-vis-NIR (near-IR) spectrophotometer. All background-corrected spectra were acquired at a scan speed of 125.00 nm/min with a 0.48 s integration time.

Elemental Analyses. Elemental analyses were performed using a ThermoElectron X-series inductively coupled plasma mass spectrometer. Samples (100 µL of 100 mg/mL BSA or a BSA-SWNT dispersion) were acid-digested using a protocol developed in association with PreciLab (Addison, TX).7 All samples and standard solutions were sprayed into flowing argon and passed into the torch which was inductively heated to ∼10 000 °C. Mo was calibrated using blank, 250, 1000, and 5000 ppt standard solutions, and Co was calibrated using blank, 50, 100, and 250 ppt standard solutions. SDS-PAGE for Quantification of SWNTs. Standard SDS-PAGE14 was performed using a Hoefer mini vertical gel caster for 10 cm × 8 cm plates with 1.5 mm thick spacers and 10 well combs. Polyacrylamide gels were prepared from a 40% stock solution that had a ratio of acrylamide to bisacrylamide of 29:1. Each gel had two layers: a larger pore stacking gel (4% polyacrylamide, pH 6.8) cast over a smaller pore resolving gel (10% polyacrylamide, pH 8.8). Samples were mixed with 2× SDS sample loading buffer to a final concentration of 2% SDS and 5% 2-mercaptoethanol and boiled for 2 min to reduce the disulfide bonds in proteins. Samples were subsequently loaded into the wells of the stacking gel, and an electric current was applied at a constant 100 V for 2 h. Following electrophoresis, optical images of gels were acquired using a 16-bit flatbed scanner (Visioneer 9520 photo scanner) at a resolution of 1000 dpi. In some cases, protein bands in the resolving gel were stained with 0.25% (w/v) coommassie brilliant blue dissolved in a 40% methanol/10% acetic acid solution. Routine quantitation of digitized gel bands was performed using the scanner and ImageQuant 5.2 software. All sample gel band intensities were background-subtracted using the intensities from a control gel band that did not contain SWNTs. The analysis of all SWNT-containing band intensities obtained by the SDS-PAGE method used a calibration curve prepared with our standard BSA-SWNT suspension. In brief, the percentage of SWNTs by weight in the starting SWNT powder was estimated as 77% based on our thermogravimetric analyses (TGA),6 which closely matched the weight percent reported by the SWNT manufacturer. Thus, our standard BSA-SWNT suspension prepared with 1.0 mg of powder in 1.0 mL of BSA was taken to contain 0.77 mg/mL SWNTs. Confocal MicroRaman Spectroscopy. All Raman spectra acquisition and sample preparation methods were similar to those described previously by Chin et al.15 Spectra were acquired with a Jobin Yvon Horiba high-resolution LabRam Raman microscope system with a 250 µm entrance slit and a 1100 µm pinhole. The 633 nm laser excitation was provided by a Spectra-Physics model 127 helium-neon laser operating at 20 mW. The power density emanating from the 50×/0.5 NA LM-Plan objective was typically 3 mW as measured using a Newport model 1815C power meter with an 818UV series photodetector. Wavenumber calibration was performed using the 520.5 cm-1 line of a silicon wafer with a spectral resolution of ∼1 cm-1. The acquisition time for a 300 cm-1 spectral region was 10 s; all spectra were plotted as the average of three scans. Raman spectra of BSA-SWNT dispersions were acquired by placing them into 35 mm polylysine(14) Laemmli, U. K. Nature 1970, 227, 680–685. (15) Chin, S.-F.; Baughman, R. H.; Dalton, A. B.; Dickmann, G. R.; Draper, R. K.; Mikoryak, C.; Musselman, I. H.; Poenitzsch, V. Z.; Xie, H.; Pantano, P. Exp. Biol. Med. 2007, 232, 1236–1244.

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coated glass bottom “imaging” dishes (MatTek, Ashland, MA). Raman spectra of SDS-PAGE gel samples were acquired after the gel was dried between two sheets of semipermeable cellophane (Idea Scientific Co., Minneapolis, MN) and placed on a glass microscope slide. Cell Culture. Normal rat kidney cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10 mM HEPES, 37 mg/mL sodium bicarbonate, 4.5 mg/mL D-glucose, 0.29 mg/mL L-glutamine, and 5% (v/v) FBS in a 37 °C incubator with 90% air and 10% CO2. To determine the number of cells in a given sample, cells grown in tissue culture dishes were first detached using 0.05% (w/v) trypsin. Aliquots of these cell suspensions were diluted in an isotonic solution (Isoton II), and the numbers of cells in the solutions were measured using a Beckman Coulter particle counter (Miami, FL). Uptake of BSA-SWNTs by NRK Cells. Medium for cell uptake experiments was made from 2× concentrated and supplemented DMEM plus 20% FBS. Aliquots of this 2× medium were mixed with H2O (control), aqueous BSA solutions, or BSA-SWNT dispersions in a 1:1 ratio. Note that the final concentrations of DMEM and supplements in the control or experimental medium were the same as in the regular growth medium, that the FBS concentration was increased from 5% to 10%, and that the final concentrations of BSA solution or BSA-SWNT dispersions was reduced by half. For a typical experiment, 1-2 × 106 cells were first plated in 100 mm tissue culture dishes in regular growth medium. After 1 day of incubation, the cells were washed twice with PBS before the addition of the control or experimental media. The cells were then incubated at 37 or 4 °C for various lengths of time. The 4 °C experiments were performed by placing the dishes in a large gas tight bag filled with 10% CO2 and incubating them in a 4 °C refrigerator. Extracting SWNTs from Cells. Following incubation, cells were washed twice with DMEM (no SWNTs) and twice with PBS before being detached from the dish with trypsin. The suspended cells were collected by gentle centrifugation at 60g for 7 min and resuspended in PBS to remove traces of trypsin before a final centrifugation. Cells in the pellet were lysed by resuspending them in 200 µL of 1% SDS, 1 mM MgCl2, and 1 mM CaCl2 for 2 h in a 37 °C water bath. The cell lysate was treated with 20 µg of DNase I for 2 h at 37 °C to degrade released DNA and reduce the viscosity of the solution. In some cases, cell lysate samples were heated to 60 °C to ensure complete lysis. All cell lysate samples were stored at -20 °C. Protein Assays. The total cellular protein content in cell lysate samples was determined using a microplate BCA protein assay kit (Pierce Chemical, Rockford, IL). Aqueous BSA standards were prepared from a 2.0 mg/mL BSA stock solution. In brief, 10 µL of diluted cell lysate samples and standards were dispensed into 96-well microtiter plates followed by the addition of 200 µL of the BCA reagent. The microtiter plate was covered and incubated at 37 °C for 30 min. The plate was cooled to room temperature before the absorbance at 595 nm was measured using a Bio-Rad 680 Microplate Reader (Hercules, CA). The protein concentration of each sample was determined using the best-fit curve from the calibration plot created using the BSA standards. When performing SDS-PAGE of cell lysates, the volumes of the cell lysates 2946

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applied to each well were adjusted so that the amount of protein was the same. Control experiments demonstrated that SWNTs in the ranges used in our studies did not interfere with the BCA assays. RESULTS Characterization of BSA-SWNT Dispersions. The biocompatible dispersant chosen for this work was BSA, a water-soluble protein well-known to noncovalently disperse pristine and oxidized SWNTs.16-19 All BSA-SWNT dispersions were prepared using a sonication and centrifugation protocol based on the work of O’Connell et al.20 BSA-SWNT dispersions prepared using this protocol yielded dark solutions of SWNTs that were homogeneous in appearance (Figure 1A). The protocol’s main advantage for our work is the facile production of individually dispersed SWNTs with short lengthssin our case, 100-500 nm long BSA-SWNTs, based on previous atomic force microscopy (AFM) analyses of CoMoCAT SWNTs using the same sonication/centrifugation protocol.7 Absorption Spectroscopy. UV-vis-NIR spectroscopy was also used to characterize BSA-SWNT dispersions since the observation of sharp van Hove peaks is indicative of aqueous solutions containing debundled, individually dispersed SWNTs.17 Figure 1B shows the absorption spectra of a series of BSA-SWNT dispersions prepared with increasing concentrations of BSA and the same initial mass of CoMoCAT SWNT-containing powder. The sharp absorption features observed in each spectrum correspond to the M11, S22, and S11 optical transitions of the particular metallic and semiconducting SWNT types contained in each dispersion, with (6, 5) and (7, 5) tubes comprising ∼75% of the total semiconducting SWNT structures present. The observed spectral profiles of BSA-SWNTs were also similar to the spectra of CoMoCAT SWNTs dispersed in SDS as prepared by Lolli et al.21 and Arnold et al.,22 where the two predominant semiconducting SWNT structures present were (6, 5) and (7, 5) tubes with an average diameter of 0.8 nm. Raman Spectroscopy. Figure 2A shows a representative Raman spectrum for a CoMoCAT BSA-SWNT dispersion prepared with 100 mg/mL BSA. The spectrum shows a number of well-characterized SWNT resonances,23,24 in particular, four predominant radial breathing modes at ∼256, ∼264, ∼286, and ∼301 cm-1, the D-band at ∼1306 cm-1, and the G-band at ∼1590

(16) Edri, E.; Regev, O. Anal. Chem. 2008, 80, 4049–4054. (17) Karajanagi, S. S.; Yang, H.; Asuri, P.; Sellitto, E.; Dordick, J. S.; Kane, R. S. Langmuir 2006, 22, 1392–1395. (18) Matsuura, K.; Saito, T.; Okazaki, T.; Ohshima, S.; Yumura, M.; Iijima, S. Chem. Phys. Lett. 2006, 429, 497–502. (19) Kam, N. W. S.; Dai, H. J. Am. Chem. Soc. 2005, 127, 6021–6026. (20) O’Connell, M. J.; Bachilo, S. M.; Huffman, C. B.; Moore, V. C.; Strano, M. S.; Haroz, E. H.; Rialon, K. L.; Boul, P. J.; Noon, W. H.; Kittrell, C.; Ma, J.; Hauge, R. H.; Weisman, R. B.; Smalley, R. E. Science 2002, 297, 593–596. (21) Lolli, G.; Zhang, L.; Balzano, L.; Sakulchaicharoen, N.; Tan, Y.; Resasco, D. E. J. Phys. Chem. B 2006, 110, 2108–2115. (22) Arnold, M. S.; Guler, M. O.; Hersam, M. C.; Stupp, S. I. Langmuir 2005, 21, 4705–4709. (23) Resasco, D. E.; Herrera, J. E. Encycl. Nanosci. Nanotechnol. 2004, 10, 125– 147. (24) Dresselhaus, M. S.; Dresselhaus, G.; Jorio, A.; Souza Filho, A. G.; Saito, R. Carbon 2002, 40, 2043–2061.

Figure 1. (A) Photograph of CoMoCAT BSA-SWNT dispersions prepared without BSA and with increasing concentrations of BSA: (a) 0, (b) 12.5, (c) 25.0, (d) 50.0, (e) 100, (f) 150, and (g) 200 mg/ mL. (B) Background-corrected absorption spectra of CoMoCAT BSA-SWNTs prepared with increasing concentrations of BSA (samples b-g; 12.5-200 mg/mL). Each spectrum is the average of three individual dispersions, and the predominant semiconducting SWNT structures are denoted by their (n, m) chiral indices. There was a linear relationship between the relative abundance of (6, 5) tubes, as measured by the peak height at 995 nm, and the concentration of BSA used to prepare the BSA-SWNTs (r2 ) 0.9925).

cm-1. The CoMoCAT method of SWNT synthesis produces as many as 90% semiconducting SWNTs,25 and indeed as shown in Figure 2A, the G-/G+ line shape of the G-band of BSA-SWNTs closely matches the signature G-/G+ line shape of isolated semiconducting SWNTs.26 The [1 - (D/G)] quality factor for our CoMoCAT BSA-SWNT dispersions was 0.94 ± 0.003, where D is the area of the D-band from 1240-1370 cm-1 and G is the area of the G-band from 1510-1630 cm-1.27 Figure 2B demonstrates that the G-band area is proportional to the relative amount of SWNTs dispersed in solution and that BSA-SWNT dispersions with varying amounts of dispersed (25) SouthWest-NanoTechnologies CoMoCAT Products. http://www.swnano. com/tech/sg.php (accessed September 2008). (26) Jorio, A.; Pimenta, M. A.; Souza Filho, A. G.; Saito, R.; Dresselhaus, G.; Dresselhaus, M. S. New J. Phys. 2003, 5, 139.1139.17. (27) Swan, A. In Measurement Issues in Single Wall Carbon Nanotubes; NIST Recommended Practice Guide, Special Publication 960-19; Freiman, S., Hooker, S., Migler, K., Arepalli, S., Eds.; NIST: Gaithersburg, MD, 2008; pp 36-54.

Figure 2. (A) Background-corrected Raman spectra (633 nm laser excitation) of a CoMoCAT BSA-SWNT dispersion prepared with 100 mg/mL BSA showing the radial breathing modes (inset), the D-band at ∼1306 cm-1, and the G-band at ∼1590 cm-1. (B) Plot of the linear relationship between integrated G-band intensities (1510-1630 cm-1) and the concentration of BSA used to prepare the BSA-SWNTs (r2 ) 0.9341). Each data point is the mean of five individual dispersions; the error bars show standard deviations.

SWNTs can be prepared by adjusting the BSA concentration. Unless noted otherwise, 100 mg/mL BSA was used to prepare BSA-SWNT dispersions for all cell studies. Elemental Analyses. It is critical to perform elemental analyses on dispersed SWNT samples since it is well-known that metals can affect the health of living cells. The predominant metals observed in individual 100 mg/mL BSA-SWNT dispersions were Mo (10 ppm) and Co (1.5 ppm). The observed Mo levels were below the 90 ppm EC50 (half-maximal effective concentration) of mammalian stem cells exposed to 30 nm MoO3 particles,28 and the observed Co levels were below the 19 ppm IC50 (halfmaximal inhibitory concentration) of murine fibroblasts exposed to CoCl2.29 These metal levels also indicate that g99.98% (28) Braydich-Stolle, L.; Hussain, S.; Schlager, J. J.; Hofmann, M.-C. Toxicol. Sci. 2005, 88, 412–419. (29) Sauvant, M. P.; Pepin, D.; Bohatier, J.; Groliere, C. A.; Guillot, J. Ecotoxicol. Environ. Saf. 1997, 37, 131–140.

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of the Mo and Co present in the as-received SWNT powder was not detected in BSA-SWNT dispersions, evidence that the sonication/centrifugation protocol used in preparing the dispersions is effective in removing heaver impurities such as metal-containing SWNTs and bundles. Quantitation of SWNTs in BSA-SWNT Dispersions by SDS-PAGE. We initially performed SDS-PAGE on BSA-SWNT dispersions to assess the amount of BSA protein in the dispersions. Inspection of the gels before staining for protein revealed a sharp black band at the interface between the sample loading well and the stacking gel suggesting that SWNTs were trapped at this interface. To better characterize the BSA-SWNT dispersion system, we determined what effect increasing BSA concentrations had on the amount of SWNTs dispersed. SWNT samples dispersed in increasing concentrations of BSA were electrophoresed, a digitized image of the gel was made with a flatbed scanner, and the band intensities were assessed by measuring pixel values. Band intensities were proportional to SWNT amounts by visual inspection (Figure 3A) and verified by quantitative analysis of pixel intensities (Figure 3B). When the gels were stained with coommassie blue to detect BSA, the protein was evident in the resolving gel and proportional to the concentration of BSA applied (Figure 3C). This established that the amount of SWNTs dispersed was proportional to the amount of BSA and provided quantitative information on what concentrations of BSA and SWNTs to use in further experiments. Interestingly, there appeared to be no coommassie stain associated with the SWNTs, suggesting that BSA originally on the SWNTs was stripped off by the electrophoretic procedure. To verify that the black band in the gel contained SWNTs, the gel was analyzed by microprobe Raman spectroscopy to see if distinctive Raman signatures of SWNTs were present. In these experiments, a 633 nm laser beam was focused in the middle of the vertical lane at different distances from the center of the dark band and the intensity of the SWNT G-line at ∼1590 cm-1 was plotted as a function of location in the gel. The only location in the gel where a SWNT G-line was detected coincided exactly with the position of the dark band, and no signal was evident either above or below the band (Figure 4A). The averaged Raman spectra between 1200 and 1800 cm-1 with the laser centered on the dark band is shown in Figure 4B and displays a signature G-/G+ line shape characteristic for semiconducting SWNTs. We conclude from this that SWNTs are present in the dark band at the top of the stacking gel and are not found elsewhere in the gel. The method for preparing the BSA-SWNT dispersions used in Figures 1-4 involved centrifugation of the dispersion, which removes a significant amount of material originally in the dry SWNT powder. To reproducibly prepare a standard SWNT solution that did not involve selective removal of material by centrifugation, we dispersed 1.0 mg of the dry SWNT powder by sonication in 1.0 mL of 100 mg/mL BSA solution and did no further centrifugation. The digitized band intensities from this BSA-SWNT standard suspension were corrected by the weight percentage of SWNTs (77%) and served as a standard curve for estimating the concentration of SWNTs in dispersions that were centrifuged. When different amounts of BSA-SWNTs from this 2948

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Figure 3. Optical detection of SWNTs in BSA-SWNT dispersions by SDS-PAGE. The 1 µL aliquots of BSA-SWNTs, BSA controls, or molecular weight markers were loaded onto a 4% stacking-10% resolving gel and electrophoresed at 100 V for 2 h. (A) The scanned image shows that SWNT-containing materials collected at the interface of the stacking gel and the loading wells as thin dark bands. Lane 1 contains the prestained protein molecular weight markers, lane 8 is blank, and lanes 2-7 are BSA-SWNT dispersions prepared using increasing concentrations of BSA: (lane 2) 12.5, (lane 3) 25.0, (lane 4) 50.0, (lane 5) 100, (lane 6) 150, and (lane 7) 200 mg/mL. (B) Plot of the linear relationship between the pixel intensities of the blank-corrected digitized gel bands acquired from BSA-SWNT dispersions (shown in panel A, lanes 2-7, no coommassie blue staining) and the concentration of BSA used to prepare the BSA-SWNTs (r2 ) 0.9436). Each data point is the mean of three individual dispersions. (C) The scanned image shows the 67 kDa BSA band in the resolving gel after staining with 0.5% coommassie blue. Lanes 1-8 are identical to those reported in panel A.

standard suspension were electrophoresed and quantified, the corrected optical band intensity was linear up to 575 ng of SWNTs applied to the gel (Figure 5A). In separate experiments with less material, the typical lower limit of detection of SWNTs in the gel was 5 ng (Figure 5A, inset). With the use of this SWNT calibration curve, the concentrations of SWNTs in three independently prepared and pooled BSA-SWNT dispersions (Figure 5B) were 186 ± 11, 209 ± 18, and 193 ± 17 µg/mL, an average of 196 ± 12 µg/mL. The applicability of the method was evaluated by analysis of CoMoCAT SWNTs dispersed using an aqueous 0.15% (w/w) sodium dodecylbenzenesulfonate (SDDBS) solution instead of

Figure 4. Microprobe Raman spectroscopic analysis of an SDS-PAGE lane (no coommassie blue staining) loaded with 2 µL of BSA-SWNTs following electrophoresis at 100 V for 2 h. (A) Raman spectra were acquired using 633 nm laser excitation and a 50× objective from various positions above and below the interface of the stacking gel and the loading well (LW). The plot of integrated Raman intensities from 1510 to 1630 cm-1, representing the SWNT G-resonance, as a function of vertical distance along the middle of the gel lane indicates that SWNTs are located in the dark band and are not found elsewhere in the gel. (B) Representative background-corrected Raman spectra acquired from the center of the dark band in panel A.

BSA. As shown in Figure 5A, the slopes of the standard BSA-SWNT and SDDBS-SWNT responses were similar. This is significant in that it demonstrates that the choice of dispersant does not affect the quantitation of SWNTs, which indicates that the SDS-PAGE method should find wide utility for detecting SWNTs in samples prepared using a variety of dispersants.

Uptake of BSA-SWNTs by NRK Cells and Extraction of SWNTs from NRK Cells. To determine the quantity of SWNTs taken into cells as a function of time, we prepared a dispersion diluted in tissue culture media (as described in the Experimental Section) that contained 50 mg/mL BSA and 98 µg/mL SWNT. At these concentrations, neither BSA nor SWNTs were toxic to NRK cells. For our cell uptake experiments, NRK cells were incubated in this BSA-SWNTs-containing media continuously for 1, 2, or 3 days. Subsequently, the cells were washed to remove external SWNTs and lysed with SDS, which is a widely used detergent for dissolving cellular material. Aliquots of these cell extracts were subjected to the SDS-PAGE method, the SWNT bands were digitized, and the concentration of SWNTs was determined using our standard BSA-SWNT calibration curve. The SWNTs were taken up by NRK cells as a linear function of incubation time, and there was no appreciable signal from control cells not exposed to BSA-SWNTs (Figure 6, part A and B). Microprobe Raman spectroscopy verified the presence of SWNTs in the cell lysate gel band by the strong G-line signature characteristic of semiconducting SWNTs (Figure 6C). Since we know the amount of protein applied to each lane of the gel, and that 106 cells contain 94.2 ± 6.5 µg of protein, NRK cells at 37 °C accumulated SWNTs at a rate of 30 fg/day/cell upon exposure to 98 µg/mL SWNTs. We also measured the uptake of BSA-SWNTs as a function of concentration in the medium. NRK cells were incubated continuously for 3 days in media containing 25, 49, 74, and 98 µg/mL SWNTs. SWNT uptake increased linearly with concentration, and there was no appreciable signal for control cells not exposed to BSA-SWNTs (Figure 7). The linear increase in cellassociated SWNTs as a function of time and concentration are characteristics of fluid-phase endocytosis, suggesting that the SWNTs enter cells by this process. Fluid-phase endocytosis dramatically slows at 4 °C, so we measured the uptake of BSA-SWNTs by cells at low temperature. NRK cells in culture were incubated continuously for 1 day with medium containing a constant concentration of 98 µg/mL BSA-SWNTs at either 4 or 37 °C, and the cell-associated SWNTs were measured by SDS-PAGE. There was no SWNT signal above background (compared to controls) at the low temperature, and obvious uptake at 37 °C, consistent with the possibility that the BSA-SWNTs are entering cells by fluid-phase endocytosis (Figure 8). The absence of a signal from cells that were incubated at 4 °C also suggests that there is no strong binding of BSA-SWNTs to the cell surface, evidence that entry of SWNTs into cells is not a receptor-mediated event. DISCUSSION We describe here a sensitive, reliable, and inexpensive method to quantify SWNTs in aqueous dispersions. As a model system to develop the method, we used SWNTs dispersed in the common protein BSA, which is known to effectively debundle SWNTs, wrapping them noncovalently with a biocompatible protein coat.16-19 When BSA-SWNT dispersions were subjected to SDS-PAGE, BSA passed through the stacking gel, entered the resolving gel, and migrated toward the anode as expected for a protein of molecular weight 67 kDa that had bound negatively charged SDS. The SWNTs, however, concentrated and accumulated in a sharp band at the interface between the sample loading well and the Analytical Chemistry, Vol. 81, No. 8, April 15, 2009

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Figure 5. (A) Estimate of absolute SWNT concentrations in CoMoCAT SWNT standard (STD) suspensions. The BSA-SWNT calibration standard was prepared by sonicating 1.0 mg of SWNT powder with 1.0 mL of 100 mg/mL BSA for 10 min. The resulting suspension was subjected to no subsequent centrifugation steps and was corrected by the weight percent of SWNTs found in the SWNT powder. The preparation of the SDDBS-SWNT calibration standard was identical except that 1.0 mL of 0.15% (w/w) SDDBS was used in place of BSA. Both SWNT calibration standards were diluted 20fold in SDS sample loading buffer, and various volumes were dispensed into separate SDS-PAGE loading wells. Following electrophoresis at 100 V for 2 h, the pixel intensities from the scanned image of the dark gel bands were corrected with the pixel intensities imaged from a blank gel lane. Mean and standard deviations were calculated from four independent experiments; the relationship between corrected intensities and SWNT mass was linear for BSA-SWNT and SDDBS-SWNT standards (r2 ) 0.9825 and 0.9850, respectively). Inset: expanded view of a BSA-SWNT standard curve, where each data point represents the mean obtained from three individual dispersions and the error bars represent standard deviations. Typical detection limits were in the range of 1-5 ng SWNTs per well. (B) Determination of absolute SWNT concentrations in BSA-SWNT dispersions prepared using 100 mg/mL BSA. Three different samples (X, Y, and Z) of BSA-SWNTs (each prepared by combining n g 12 individual BSA-SWNT dispersions) were diluted 10-fold, and appropriate aliquots were subjected to electrophoresis together with the standard BSA-SWNT suspension in the same gel. Following electrophoresis at 100 V for 2 h, the corrected mean pixel intensities from the scanned image of the dark gel bands were used with the BSA-SWNT calibration curve (shown in panel A) to estimate the unknown SWNT concentrations in the three BSA-SWNT samples. Each data point represents the mean obtained from three independent trials, and the error bars represent standard deviations. 2950

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Figure 6. Determination of the amount of SWNTs extracted from NRK cells cultured in media containing BSA-SWNTs as a function of incubation time. Cell lysate samples were prepared from cells incubated for 1, 2, or 3 days in media with or without BSA-SWNTs containing 98 µg/mL SWNTs. Cell lysate samples equivalent to 300 µg of total cellular protein were analyzed to ensure that equal numbers of cells were compared. (A) Scanned digital image of the control lane and the SWNT-containing bands at the top of the stacking gel following SDS-PAGE at 100 V for 2 h. Lane 1, 3 day incubation in media containing no SWNT dispersion; lanes 2-4, incubation in media containing BSA-SWNTs for 1, 2, or 3 days, respectively. (B) Plot of the SWNT content in cell lysate samples determined from corrected mean pixel intensities of the SWNT-containing bands as a function of incubation time (r2 ) 0.9922). Each data point is the mean obtained from three independent trials, and the error bars show standard deviations. (C) Microprobe Raman spectroscopic analysis of gel lanes 1, 2, and 4 shown in part A. Each background-corrected Raman spectra was an average of three spectra acquired with a 50× objective and 633 nm laser excitation focused on the interface of the stacking gel and the loading well in each lane. The appearance of the peak at ∼1450 cm-1 can be attributed to cellular lipids.

stacking gel. Moreover, we detected no BSA in this band by coommassie blue staining. These observations are consistent with

Figure 7. Determination of the amount of SWNTs extracted from NRK cells cultured in media containing various concentrations of BSA-SWNTs. Cell lysate samples were prepared from cells incubated for 3 days in media with or without BSA-SWNTs. All samples analyzed contained 300 µg of total cellular protein. (A) Scanned digital image of the control lane and the dark SWNT-containing bands at the interface of the stacking gel and the loading wells following SDS-PAGE at 100 V for 2 h. Lane 1, 3 days of incubation in media containing no SWNT dispersion: lanes 2-5, 3 days of incubation in media containing BSA-SWNTs with SWNT concentrations of 25, 49, 74, and 98 µg/mL, respectively. (B) Plot of the SWNT content in cell lysate samples determined from corrected mean pixel intensities of the SWNT-containing bands as a function of SWNT concentration (r2 ) 0.9811). Each data point is the mean obtained from three independent trials, and the error bars show the standard deviations.

the idea that the SDS stripped the BSA off the SWNTs and also replaced the BSA as the coat on the SWNTs. Thus, the SWNTs acquired a negative charge from the SDS coat and also migrated toward the anode. Upon reaching the stacking gel, the SDS-SWNT complexes are apparently too large to pass into the gel and so accumulate at the gel surface in a sharp band. We demonstrated that this gel band contained SWNTs by microprobe Raman analyses and that the intensity of the band was proportional to the amount of SWNTs loaded onto the gel. Two features of this method suggest that it should be especially useful in quantifying SWNTs in biological samples. First, the SWNTs initially dispersed in the volume of fluid that is applied to the sample loading well are efficiently concentrated at the stacking gel surface. Thus, dilute SWNT dispersions, as often found with biological samples, are concentrated to enhance the sensitivity of detection. Second, the SDS will likely strip off and replace most endogenous proteins present in biological fluids that may bind SWNTs. Further, most of these proteins will migrate into the stacking gel, separating from the SWNT band, so they will not interfere with detection of the SWNTs.

Figure 8. Determination of the amount of SWNTs extracted from NRK cells cultured at 4 or 37 °C in media containing BSA-SWNTs. Cell lysate samples were prepared from cells incubated for 1 day at 4 or 37 °C in media with BSA or BSA-SWNTs. All samples analyzed contained 300 µg of total cellular protein. (A) Scanned digital image of the gel area at the interface of the stacking gel and the loading wells following SDS-PAGE at 100 V for 2 h. Lane 1, 1 day of incubation at 37 °C in media containing neither BSA nor BSA-SWNTs: lane 2, 1 day of incubation at 37 °C in media containing BSA; lanes 3 and 4, cells incubated in media containing 98 µg/mL BSA-SWNTs at 4 and 37 °C, respectively. (B) Plot of the SWNT content in cell lysate samples determined from corrected mean pixel intensities of the SWNT-containing bands. Each data point is the mean from three independent trials, and the error bars show the standard deviations.

To test the method with a model biological sample, NRK cells in culture were exposed to medium containing BSA-SWNTs for various times, the cells were lysed with SDS, and the extracts were subjected to SDS-PAGE. A SWNT band was clearly present at the stacking gel surface in cells exposed to SWNTs, the band contained SWNTs as evidenced by the characteristic Raman G-band signature of SWNTs, and the intensity of the band increased as a linear function of incubation time. The process of fluid-phase endocytosis is characterized by a linear uptake of material,30,31 suggesting that the BSA-SWNTs may enter cells by this type of endocytic process. Two other characteristics of fluid-phase uptake are that the process dramatically slows at 4 °C and that uptake is a linear function of concentration.30,31 We observed both of these features for the uptake of BSA-SWNTs. Thus, the evidence is consistent with the proposal that BSA-SWNTs enter NRK cells by fluid-phase endocytosis. Although further study is needed to understand how SWNTs prepared in different ways enter cells, we7 and others8,11,19,32 have observed SWNT accumulation in intracellular vesicles after energy-dependent uptake. (30) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37–44. (31) Silverstein, S. C.; Steinman, R. M.; Cohn, Z. A. Annu. Rev. Biochem. 1977, 46, 669–722. (32) Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600–11605.

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CONCLUSION We have developed a rapid, sensitive, and inexpensive method for detecting and quantifying SWNTs in aqueous solutions. The method is particularly useful for assessing SWNTs in biological extracts, and we used the approach to study parameters of BSA-SWNT uptake by cultured cells. The results suggest that BSA-SWNTs enter NRK cells by fluid-phase endocytosis. In preliminary experiments, we have also successfully applied the method to detecting carboxylated SWNTs and to measuring SWNTs in blood and tissue extracts, which will be useful for biodistribution and pharmacokinetic studies of SWNTs in intact animals.

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ACKNOWLEDGMENT This work was supported by Grants from the Robert A. Welch Foundation (P.P.; Grant AT-1364), the Department of Defense (TATRC), The State of Texas Norman Hackerman Advanced Research Program (R.K.D.), and by funds from the UT-Dallas Center for Applied Biology (R.K.D.). The authors are grateful for contributions to this work from Don Gray, Kate Walker, and Matt Wallack, and John Bonds.

Received for review November 24, 2008. Accepted February 19, 2009. AC802485N